DRG11-responsive axonal guidance and outgrowth of neurite (dragon) gene family

ABSTRACT

This invention features methods and compositions useful for treating and diagnosing diseases of the nervous system, retina, skin, muscle, joint, and cartilage using a Dragon family protein. Protein and nucleic acid sequences of human, murine, zebrafish, and  C. elegans  Dragon family members are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.11/230,180, filed Sep. 19, 2005, which is a divisional of U.S.application Ser. No. 10/419,296, filed Apr. 17, 2003, which claimsbenefit of U.S. Provisional Application No. 60/373,519, filed Apr. 18,2002, each of which is hereby incorporated by reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant No.5R01-NS038253 awarded by the National Institutes of Health. TheGovernment has certain rights in this invention.

FIELD OF THE INVENTION

This invention relates to a DRG 11-responsive gene and its homologsuseful for treating and diagnosing diseases, developmental defects, andinjuries of the nervous system, retina, skin, muscle, bone, and jointtissue.

BACKGROUND OF THE INVENTION

Developmentally regulated transcription factors drive developmental geneprograms that result in embryo formation and the birth, proliferation,growth, migration, and differentiation of the cells that eventually makeup the different tissues of the body. This involves the expression andrepression of many genes including those whose protein products act asregulators of this process as signal molecules. When the signal proteinsare secreted, they may act both as paracrine signals between differentcells, including on stem cells, and as autocrine signals on the samecells that produce the signal molecule. When the protein is notsecreted, but rather inserted into the cell membrane, it may contributeto cell-cell interactions.

In the case of the developing nervous system, multiple secreted andnon-secreted signal molecules expressed at different times and indifferent spatial locations are involved in: (i) determining theinduction of the neural plate; (ii) regionalization of the neural tubealong dorsoventral and anteroposterior axes; (iii) generation of neuronsand glia from multipotent precursors (neuronal determination); (iv)determination of survival or apoptotic cell death; (v) migration ofneurons; (vi) differentiation and regional patterning of neurons; (vii)neurite outgrowth and axon guidance; (viii) formation of specificsynaptic connections between neurons, and (ix) determiningneuronal-glial interactions.

Some of these signal molecules may be re-expressed in the adult afterinjury, or the failure of such re-expression may relate to the failureof mature neurons to survive, grow, or regenerate after injury. Some ofthe signal molecules may act in pathological situations to eitherpromote or suppress abnormal growth or function. These signal molecules,acting on specific transmembrane receptors, may serve as neuronaldeterminants, survival factors, growth factors, guidance cues, ordifferentiation factors, and many may have potential therapeutic rolesas biological agents beyond their specific involvement in development.Such factors can have biological activity both in vivo and formaintaining cultured cells in vitro, or for converting pluripotent stemcells into specific neuronal or non-neuronal subtypes. Similarly,mimicking the action of these signal molecules by activating theirmembrane bound receptors or the intracellular signal transductionpathways coupled to their receptors, may also have therapeuticpotential.

SUMMARY OF THE INVENTION

We have discovered a novel gene family, designated “Dragon”(DRG11-Responsive Axonal Guidance and Outgrowth of Neurite), expressedin the nervous system, retina, skin, muscle, bone, and joint tissue.Three homologous proteins have been identified in each of the mouse,zebrafish, and human. A partial sequence of an ortholog has also beenidentified in C. elegans.

The invention features substantially pure DRAGON, Dragon-like 1 (DL-1),and Dragon-like 2 (DL-2) proteins, fragments, homologs, and orthologs,as well as non-naturally occurring but substantially identical proteins.Preferably, the proteins are mammalian and/or are substantiallyidentical to murine DRAGON, Dragon-like 1 (mDL-1), or Dragon-like 2(mDL-2) (SEQ ID NO: 5-7, respectively), or human DRAGON, Dragon-like 1(hDL-1), or Dragon-like 2 (hDL-2) (SEQ ID NO: 8-10, respectively). Alsoincluded in this invention are the zebrafish homologs of DRAGON, DL-1,and DL2 (SEQ ID NO: 28-30, respectively) and the C. elegans homologcontaining the polypeptide sequence of SEQ ID NO: 18.

Also featured are substantially pure nucleic acids which encode DRAGONand the Dragon-like proteins, for example, from mammals. Preferably, thenucleic acids are substantially identical to the murine DRAGON, DL-1, orDL2 (SEQ ID NO: 1-3, respectively), human DRAGON, DL-1, or DL-2 (SEQ IDNO: 4, 31, and 32, respectively), or zebrafish DRAGON, DL-1, or DL-2(SEQ ID NO: 25-27, respectively). Other embodiments include nucleicacids which, but for the degeneracy of the genetic code, would besubstantially identical to the identified murine, human, and zebrafishDragon family members, as well as nucleic acids which hybridize underhigh stringency or, less preferably, low stringency conditions to any ofthose nucleic acids.

Monoclonal and polyclonal antibodies that selectively bind the Dragonfamily proteins, for example, of SEQ ID NO: 5-10, 18, or 28-30, can alsobe prepared and are included in the invention. Preferably, theantibodies specific for murine DRAGON bind a protein sequence encoded byresidues 38-56, 261-278, or 369-386 of SEQ ID NO: 5, and the antibodiesspecific for hDRAGON bind a protein sequence encoded by residues 54-72,277-294, or 385-408 of SEQ ID NO: 8. Other immunogenic portions of theproteins of the Dragon family also can be used for antibody production.

Also provided are expression vectors containing a coding sequenceoperably linked to an expression control element, such as a promoter orenhancer element. Preferred coding sequences include those that encodeany Dragon family protein or fragment thereof, or express an antisensenucleic acid which is complementary to and capable of hybridizing to anucleic acid that encodes a member of the Dragon family, or itspromoter. Preferably, these antisense nucleic acids include at least 12and more preferably at least 25 contiguous nucleotides. These vectorscan be used to transfect cells, resulting in the production of Dragonfamily proteins and/or sense or antisense nucleic acids. Suitable cellsinclude, for example, bacteria, yeast, and mammalian, for example, humancells. Transfection may result in stable or transient Dragon expression.

Transgenic non-human organisms with altered Dragon expression levels arealso included in the invention. A transgenic organism of this inventioncan either have a homologous Dragon-coding sequence (for example, ahuman DRAGON-coding sequence) inserted into its genome such that Dragonexpression is increased, or the endogenous Dragon gene(s) can bedisrupted rendering the organism Dragon-deficient. Any non-humanorganism can be used for transgenic Dragon expression or can be renderedDragon-deficient. Preferably, the transgenic or Dragon-deficientorganisms are C. elegans or mammals, such as mice.

The invention also provides a method for treating a patient with aneurological disorder, a developmental deficit, or a congenital disorderof the nervous system by administering a therapeutically effectiveamount of a Dragon family protein. Preferably, the Dragon protein is amammalian protein, for example, hDRAGON or hDL-2. Neurological disordersthat can be treated according to the methods of this invention includeneurodegenerative diseases such as Parkinson's disease, Huntington'sdisease, Alzheimer's disease, motor neuron diseases and other spinalmuscular atrophies, and neuropathies including diabetic neuropathy andinherited demyelinating neuropathies. Other nervous system injuries orfunctional disorders that can be treated, for example, by DRAGON orDL-2, include those caused by trauma, (e.g., peripheral nerve, dorsalroot, spinal cord, and brain injury) cerebrovascular disease (e.g.,ischemia, thrombosis, or hemorrhage), chemical-induced neurotoxicity,metabolic diseases, infection, primary and secondary neoplasms of thenervous system, congenital abnormalities of nervous system (e.g.,neurofibromatosis, phakomatosis, cerebral palsy, mental retardation),sensory and motor-abnormalities, (e.g., pain, nociceptive inflammatory,peripheral and central neuropathic pain), cognitive and mood disorders,psychoses, epilepsy, coordination disorders. Nervous system disorderscaused by non-neuronal cells are also amenable to treatment usingmammalian DRAGON or DL-2. Disorders of this type include, for example,demyelinating disorders, axonal conduction deficits, and abnormal growthof glial cells (e.g., gliomas, Schwanomas, and neurofibromatosis),degenerative diseases of the retina, cochlea and olfactory mucosa.Treatment may be by any method and is preferably by oral, parenteral,intrathecal, or intracerebrovascular administration.

DRAGON may also be used to treat disorders of the skin. Preferably, theDRAGON protein is mammalian, most preferably human. Administration of aDRAGON protein will preferably be topical in a cream, gel, ointment, orirrigation solution. Alternatively, the DRAGON protein can beadministered by subcutaneous injection at or near the lesion site.Disorders amenable to treatment include trauma (i.e., accidental orsurgical), burns (i.e., chemical, thermal, or radiation), allergicreactions such as eczema, psoriasis, or contact dermatitis, pressureulcers, and acne.

The DRAGON protein may also be used to treat disorders of the retina andoptic nerve. Administration of a DRAGON protein, preferably a mammalian(i.e., human) protein, may be in the form of eye drops or an irrigationsolution. Alternatively, intraocular or intraorbital injection may beused. Retinal disorders amenable to treatment using a DRAGON proteininclude, for example, traumatic injuries (i.e., detached retina),macular degeneration, and sarcoidosis. Optic nerve diseases amenable totreatment with a DRAGON protein include, for example, ischemic opticneuropathy, primary glaucomatous optic nerve disease (GOND), toxic opticnerve disease, and Leber's Hereditary Optic Neuropathy (LHON).

Dragon-like 1 (DL-1) can be used for treatment of disease conditions ofthe bone, muscle, joint, or cartilage including muscle wasting,congenital myopathies, muscular dystrophy, and the innervation of muscleby motor axons (e.g., following peripheral nerve injury), bone fracture,metabolic disorders of bone, disorders of bone formation or resorption,neoplasms of bone, congenital abnormalities of bone (bone dyplasias,achondorplasia, or endochondromatosis), inflammatory or degenerativejoint diseases (e.g. arthritis, osteoarthritis, and rheumatoidarthritis), muscle paralysis and other diseases resulting in the failureof the neuromuscular system (e.g., myasthenia gravis).

DL-2 can also be used for treatment of cardiovascular diseases anddisorders including, for example, developmental heart abnormalities,congenital cardiac malformations, and blood vessel malformations (e.g.,aneurisms).

Embryonic or adult pluripotent cells can be induced to differentiateinto neuronal, retinal, epidermal (DRAGON or DL-2), or myogenic (DL-1)phenotypes by contacting the cells with a Dragon protein in a mannersufficient to increase the Dragon biological activity. The Dragonprotein that contacts the cells to induce a particular phenotype mayresult from the overexpressing a Dragon nucleic acid by the cells or thecells can be cultured in the presence of an exogenously applied Dragonprotein. Preferably, the cells are human embryonic stem cells or bonemarrow-derived stem cells. Optionally, a TGF-β family member or TGF-βreceptor can be inhibited to aid in inducing a neuronal phenotype. Cellsthat have been induced or regulated by DRAGON or DL-2 treatment may beused for subsequent administration to patients to replace lost orabnormally functioning cells.

The invention also provides a method for diagnosing a Dragon-relatedcondition in a patient. Typically, the condition is diagnosed byassessing a Dragon family nucleic acid (e.g., gene) for one or moremutations that reduce Dragon biological activity. The Dragon familynucleic acid may be, for example, DRAGON, DL-1, or DL-2. These mutationsmay be in an untranslated region such that gene expression is impaired.Alternatively, the mutation may be in a coding region, causing areduction in protein function. Common techniques for assessing Dragonnucleic acids include, for example, Northern and Southern analysis,including the polymerase chain reaction (PCR), and restriction fragmentlength polymorphism (RFLP) analysis. Any appropriate patient sample canbe used in the diagnostic screening provided; however, particularlyuseful sample sources include, for example, blood samples and tissuebiopsies.

The Dragon family proteins and nucleic acids can also be used toidentify candidate compounds which modulate (increase or decrease)Dragon expression, or mimic or inhibit Dragon biological activity.Compounds identified using these screening techniques are useful fortreating Dragon-related diseases and conditions described herein. Amethod for identifying candidate compounds that modulate Dragon activityincludes the steps of: (a) exposing a sample to a test compound, whereinthe sample contains a Dragon nucleic acid, a Dragon promoter operablylinked to a reporter gene (for example, a detectable label such asalkaline phosphatase), or a Dragon protein; and (b) identifying a usefulcandidate compound by assaying for a change in the level of Dragonexpression or biological activity in the sample, relative to a samplenot exposed to the test compound.

The invention also features a method for identifying endogenous andsynthetic Dragon family binding partners such as Dragon receptors andDragon ligands. The method includes the steps of: (i) providing a Dragonfusion protein which consists of a Dragon protein linked to a tagmolecule; (ii) contacting a sample containing a putative Dragon bindingpartner with a Dragon fusion protein under conditions which allow for aDragon-Dragon binding partner complex to form, (iii) detecting theDragon fusion protein by detecting the tag molecule, and (iv)interpreting the Dragon fusion protein detection to determine whetherthe Dragon fusion protein is complexed to a Dragon binding partner fromthe sample. In one embodiment, a further step of (v) isolating theDragon-Dragon binding partner complex using a method directed againstthe tag molecule. In one preferred embodiment, the step (iii) detectingis done using a detectably labeled antibody. Preferred techniques instep (iii) include, for example, Western blotting and ELISA assays. Inanother preferred embodiment, the step (v) isolating is done usingaffinity chromatography.

By “Dragon protein” or “Dragon-family protein” is meant any polypeptidethat is substantially identical to the human, murine, or zebrafishDRAGON, Dragon-like 1 (DL-1), or Dragon-like 2 (DL-2) proteins. Dragonproteins also include substantially identical fragments of DRAGON, DL-1,DL-2, or any other Dragon-family protein. Dragon fragments are typicallyat least 50, 100, 150, 200, 250, 300, 350, or 400 amino acids in length.

By “Dragon nucleic acid” or “Dragon-family nucleic acid” is meant anypolynucleotide that is substantially identical to the human or murineDRAGON, DL-1, or DL-2 cDNA sequences, any polynucleotide having adegenerate sequence that encodes a DRAGON, DL-1, or DL-2 protein, or anypolynucleotide whose complement hybridizes to a human or murine DRAGON,DL-1, or DL-2 sequence under high stringency conditions. Alternatively,a Dragon nucleic acid encodes a protein which is substantially identicalto the human or murine DRAGON, DL-1, or DL-2 proteins.

By “DRAGON protein” is meant a polypeptide having a sequencesubstantially identical to SEQ ID NO: 5, 8, 18, or 28.

By “DL-1 protein” is meant a polypeptide having a sequence substantiallyidentical to either SEQ ID NO: 6, 9, or 29.

By “DL-2 protein” is meant a polypeptide having a sequence substantiallyidentical to either SEQ ID NO: 7, 10, or 30.

By “DRAGON nucleic acid” is meant a polynucleotide having a sequencewhich encodes a DRAGON protein. Preferably, a DRAGON nucleic acid issubstantially identical or hybridizes under high stringency conditionsto SEQ ID NO: 1, 4, or 25.

By “DL-1 nucleic acid” is meant a polynucleotide having a sequence whichencodes a DL-1 protein. Preferably, a DL-1 nucleic acid is substantiallyidentical or hybridizes under high stringency conditions to SEQ ID NO:2, 26, or 31.

By “DL-2 nucleic acid” is meant a polynucleotide having a sequence whichencodes a DL-2 protein. Preferably, a DL-2 nucleic acid is substantiallyidentical or hybridizes under high stringency conditions to SEQ ID NO:3, 27, or 32.

By “substantially identical” is meant a polypeptide or nucleic acidexhibiting at least 50%, 75%, 85%, 90%, 95%, or even 99% identity to areference amino acid or nucleic acid sequence. For polypeptides, thelength of comparison sequences will generally be at least 20 aminoacids, preferably at least 30 amino acids, more preferably at least 40amino acids, and most preferably 50 amino acids, or full-length. Fornucleic acids, the length of comparison sequences will generally be atleast 60 nucleotides, preferably at least 90 nucleotides, and morepreferably at least 120 nucleotides, or full length.

By “high stringency conditions” is meant any set of conditions that arecharacterized by high temperature and low ionic strength and allowhybridization comparable with those resulting from the use of a DNAprobe of at least 40 nucleotides in length, in a buffer containing 0.5 MNaHPO₄, pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA (Fraction V), at atemperature of 65° C., or a buffer containing 48% formamide, 4.8×SSC,0.2 M Tris-Cl, pH 7.6, 1× Denhardt's solution, 10% dextran sulfate, and0.1% SDS, at a temperature of 42° C. Other conditions for highstringency hybridization, such as for PCR, Northern, Southern, or insitu hybridization, DNA sequencing, etc., are well-known by thoseskilled in the art of molecular biology. See, e.g., Ausubel et al.,Current Protocols in Molecular Biology, John Wiley & Sons, New York,N.Y., 2000, hereby incorporated by reference.

By “Dragon antisense nucleic acid” is meant a nucleic acid complementaryto a Dragon coding, regulatory, or promoter sequence, including humanand murine DRAGON, Dragon-like 1 (DL-1) and Dragon-like 2 (DL-2).Preferably, the antisense nucleic acid decreases expression (e.g.,transcription and/or translation) of the Dragon by at least 5%, 10%,25%, 50%, 75%, 90%, 95%, or even 99%. Preferably, the Dragon antisensenucleic acid comprises from about 8 to 30 nucleotides. A Dragonantisense nucleic acid may also contain at least 40, 60, 85, 120, ormore consecutive nucleotides that are complementary to a Dragon mRNA orDNA, and may be as long as a full-length Dragon gene or mRNA. Theantisense nucleic acid may contain a modified backbone, for example,phosphorothioate, phosphorodithioate, or other modified backbones knownin the art, or may contain non-natural internucleoside linkages.

A Dragon antisense nucleic acid may also be encoded by a vector wherethe vector is capable of directing expression of the antisense nucleicacid. This vector may be inserted into a cell using methods known tothose skilled in the art. For example, a full length Dragon nucleic acidsequence, or portions thereof, can be cloned into a retroviral vectorand driven from its endogenous promoter or from the retroviral longterminal repeat or from a promoter specific for the target cell type ofinterest. Other viral vectors which can be used include adenovirus,adeno-associated virus, vaccinia virus, bovine papilloma virus, or aherpes virus, such as Epstein-Barr Virus.

By “vector” is meant a DNA molecule, usually derived from a plasmid orbacteriophage, into which fragments of DNA may be inserted or cloned. Avector will contain one or more unique restriction sites, and may becapable of autonomous replication in a defined host or vehicle organismsuch that the cloned sequence is reproducible. A vector contains apromoter operably linked to a gene or coding region such that, upontransfection into a recipient cell, an RNA is expressed.

By “substantially pure” is meant a nucleic acid, polypeptide, or othermolecule that has been separated from the components that naturallyaccompany it. Typically, the polypeptide is substantially pure when itis at least 60%, 70%, 80%, 90% 95%, or even 99%, by weight, free fromthe proteins and naturally-occurring organic molecules with which it isnaturally associated. For example, a substantially pure polypeptide maybe obtained by extraction from a natural source, by expression of arecombinant nucleic acid in a cell that does not normally express thatprotein, or by chemical synthesis.

By a “promoter” is meant a nucleic acid sequence sufficient to directtranscription of a gene. Also included in the invention are thosepromoter elements which are sufficient to render promoter-dependent geneexpression controllable for cell type-specific, tissue-specific orinducible by external signals or agents (e.g. enhancers or repressors);such elements may be located in the 5′ or 3′ regions of the native gene,or within an intron.

By “operably linked” is meant that a nucleic acid molecule and one ormore regulatory sequences (e.g., a promoter) are connected in such a wayas to permit expression and/or secretion of the product (e.g., aprotein) of the nucleic acid molecule when the appropriate molecules(e.g., transcriptional activator proteins) are bound to the regulatorysequences.

By “signal sequence” is meant a nucleic acid sequence which, whenoperably linked to a nucleic acid molecule, facilitates secretion of theproduct of the nucleic acid molecule. The signal sequence is preferablylocated 5′ to the nucleic acid molecule.

By “transgene” is meant any piece of nucleic acid that is inserted byartifice into a cell, or an ancestor thereof, and becomes part of thegenome of the animal which develops from that cell. Such a transgene mayinclude a gene which is partly or entirely heterologous to thetransgenic animal, or may represent a gene homologous to an endogenousgene of the animal.

By “transgenic” is meant any cell which includes a nucleic acid sequencethat has been inserted by artifice into a cell, or an ancestor thereof,and becomes part of the genome of the organism which develops from thatcell. Preferably, the transgenic organisms are transgenic mammals (e.g.,rodents or ruminants), or C. elegans, Zebra fish, or Drosophila.Preferably the nucleic acid (transgene) is inserted by artifice into thenuclear genome.

By “antibody that selectively binds” is meant an antibody capable of ahigh affinity interaction with a specific target molecule, having adissociation constant of <1 μM, <100 nM, <10 nM, <1 nM, or even <100 pM.Preferably, the antibody has at least 10-fold, 100-fold, 1,000-fold, oreven 10,000-fold lower affinity for other, non-target molecules.

By a “neurological disorder” is meant any disease or condition thatcauses injury to any component of the peripheral or central nervoussystem, including the retina. Neurological disorders include acute andchronic conditions. Acute conditions include, for example, trauma,stroke, and chemical-induced neurotoxicity. Chronic conditions include,for example, neurodegenerative diseases and cancers of the nervoussystem including gliomas, schwanomas, and astrocytomas. Neurologicaldisorders can also arise from developmental defects, including inheritedor congenital defects (e.g. cerebral palsy), and autoimmune diseases(e.g., multiple sclerosis). Neurological disorders also includefunctional disorders such as paralysis, and epilepsy, as well assensory, mood, and psychomotor disorders (e.g., fibromyalgia,dysthesia).

By a “neurodegenerative disease” is meant any disease of the central,peripheral, or autonomic nervous system that is characterized byprogressive neuronal loss or dysfunction, including but not limited toAlzheimer's Disease, dementia pugilistica, Parkinson's Disease,Huntington's Disease, Niemann-Pick disease, multiple sclerosis,neuropathies (e.g., central, peripheral, compression type, and diabetic)and ischemic conditions such as stroke and cerebral artery infarction.Defects in myelin repair are also considered neurological diseases. Thedefects may arise during the process of demyelination, the removal ofmyelin debris following injury, or the remyelination process.

By a “bone disorder” is meant any condition of the bone which ischaracterized by altered bone remodeling. Bone disorders includephysical traumas such as bone fractures, metabolic bone diseases such asPaget's disease and hyperostosis, and bone neoplasms (e.g.,oesteochondromas, oesteogenic sarcoma).

By a “joint disorder” is meant any trauma or disease process whichcauses inflammation in or around the cartilage or joint capsule. Jointdisorders include, for example, inflammatory arthritis, rheumatoidarthritis, and osteoarthritis.

By a “muscle disorder” is meant any dysfunction of muscle tissueregardless of cause. Muscle disorder may arise for congenitalabnormalities, trauma, metabolic disease, or autoimmune disease. Muscledisorders include, for example, muscular dystrophy, myasthenia gravis,transient and periodic muscle paralysis, muscle wasting diseases,muscular dystrophy, myotonia congenital, myotonic dystrophy, and loss ofinnervation of motor endplates.

By a “Dragon-related condition” is meant any disease or disorder whichis associated with the dysfunction or altered (increased or decreased)activity or expression of any one or more of the Dragon protein family.Alternatively, Dragon-related conditions can also refer to any diseaseor disorder which, although not associated with Dragon dysfunction, isamenable to treatment by modulating (increasing or decreasing) theactivity or expression of any one or more Dragon proteins or nucleicacids or by mimicking their actions. Dragon-related conditions include,for example, neurological, retinal, and skin disorders,neurodegenerative diseases, and muscle, bone, or joint disorders.

By a “therapeutically effective amount” is meant a quantity of compound(e.g., a Dragon family protein) delivered with sufficient frequency toprovide a medical benefit to the patient. Thus, a therapeuticallyeffective amount of a Dragon family protein is an amount sufficient totreat or ameliorate a Dragon-related condition or symptoms.

By “treating” is meant administering a pharmaceutical composition forthe purpose of improving the condition of a patient by reducing,alleviating, or reversing at least one adverse effect or symptom.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A is a schematic diagram illustrating a strategy for genomicscreening with a CpG island library. The plasmid DNA was bound withGST-DRG11-DBD and passed through a nitrocellulose filter. DRG 1′-boundplasmids were eluted and amplified in bacterial cultures. TheDRG11-bound plasmids were concentrated by repeating the cycle a total offive times. FIG. 1B is the nucleic acid (SEQ ID NO: 1) and deducedpolypeptide sequence of murine DRAGON (mDRAGON; SEQ ID NO: 5). TheDRAGON protein contains an N-terminal signal peptide and a C-terminalglycophosphatidyl inositol (GPI) anchor.

FIG. 2 is a graph illustrating the result of a computationalstructure-function analysis of mDRAGON (SEQ ID NO: 5), demonstrating thepresence of a signal sequence which results in protein secretion.

FIG. 3 is a sequence alignment of hDRAGON (SEQ ID NO: 8) and a portionof the insulin-like growth factor binding protein 2 (IGFBP2; SEQ ID NO:12).

FIG. 4 is a sequence alignment of hDRAGON (SEQ ID NO: 8) and a portionof the ephrin type-B receptor 3 precursor (EPHB3; SEQ ID NO: 13).

FIG. 5 is a sequence alignment showing domain homology between mDRAGON(SEQ ID NO: 5) and portions of human Notch 3 (SEQ ID NO: 14) and murinephosphatidylinoitol-4-kinase type II beta (SEQ ID NO: 15).

FIG. 6 is a sequence alignment showing the domain homology betweenmDRAGON (SEQ ID NO: 5) and a portion of thrombospondin-1 (SEQ ID NO: 16;THR-1) and Slit2 (SEQ ID NO: 17).

FIG. 7A is a photomicrograph of an in situ hybridization study showingthat DRAGON and DRG11 mRNAs are both expressed in the dorsal rootganglion (DRG) and the spinal cord at E12.5. FIG. 7B is a bar graphshowing the DRG11-dependent enhancer activity of the DRAGON promoterfragment. FIG. 7C shows the results of a pull-down experiment usingeither GST or GST-DBD (DBD=DRG11 DNA Binding Domain). The purifiedproteins (right panel) were incubated with the DRAGON promoter fragment,and “pulled down” using glutathione sepharose. Only GST-DBD fusionprotein pulled down the promoter fragment as assessed by agarose gelelectrophoresis. FIG. 7D is a photomicrograph of an in situhybridization study demonstrating a decrease in DRAGON mRNA expressionin the DRG and the spinal cord of DRG11−/− mouse at E14.5, compared towildtype. FIG. 7E shows the result of a Northern blot analysis of DRAGONmRNA expression in adult and embryonic E14.5 tissue. FIG. 7F shows theresult of a Northern blot analysis of DRAGON mRNA expression in wholemouse embryos during development. β-actin mRNA levels were used as aloading control.

FIG. 8A is an amino acid sequence alignment of mDRAGON (SEQ ID NO: 5),mDL-2 (SEQ ID NO: 7), and mDL-1 (SEQ ID NO: 6). FIG. 8B is an amino acidsequence alignment of mDRAGON (SEQ ID NO: 5), hDRAGON (SEQ ID NO: 8),and zDRAGON (SEQ ID NO: 26).

FIGS. 9A-9L are a series of photomicrographs showing, by in situhybridization, the developmental distribution of DRAGON family membersin the mouse embryo. FIG. 9A-9C demonstrate that DRAGON and DL-2, butnot DL-1, mRNA is expressed in mouse embryonic E14.5 spinal cord. DRAGONis the only family member expressed in the DRG. FIGS. 9D-9F aretransverse sections of whole mouse E17.5 embryo demonstrating DRAGON,DL-1, and DL-2 mRNA expression. FIGS. 9G-9I demonstrate DRAGON mRNAexpression in transverse sections of mouse E17.5 embryo head. (FIG. 9G:Mes.: mesencephalic vesicle; E.: ependymal layer; M.: mantle layer. FIG.9H: M.: myelencephalon; D: diencephalon; S: striatum; C: cortex. FIG.9I: D: DRG; S.C.: spinal cord; C.: cochlea; R.: retina; Olf.: futureolfactory lobe) FIGS. 9J-9L demonstrate DL-2 mRNA expression intransverse section of mouse E17.5 embryo head.

FIG. 10 is a series of photomicrographs showing the distribution ofDRAGON mRNA in the adult rat DRG by in situ hybridization.

FIG. 11 is a series of photomicrographs showing the distribution ofDRAGON mRNA in the brain of an E18 mouse by in situ hybridization.

FIG. 12 is a series of photomicrographs showing the distribution of DL-2mRNA in the brain of an E18 mouse by in situ hybridization.

FIGS. 13A-13D provide experimental results using a novel anti-DRAGONrabbit polyclonal antibody. FIG. 13A is a Western blot analysis ofprotein extract from untransfected HEK293 cells (−), or thosetransfected (+) with DRAGON expression vector. A distinct band having amolecular weight of about 50 KDa is recognized by the anti-DRAGONantibody in transfected, but not control, cells. ERK protein level wasused as a loading control. FIG. 13B is a photomicrograph of animmunocytochemical study showing significant staining ofDRAGON-expressing HEK cells (top). Pretreatment of DRAGON-expressing HEKcells with PI-PLC causes a significant reduction of anti-DRAGON staining(bottom). Non-transfected HEK cells show no anti-DRAGON staining (notshown). FIG. 13C is a photomicrograph of a Western blot analysis ofsamples of DRAGON-expressing HEK cell culture medium, with or withoutpretreatment using PI-PLC. A band corresponding to DRAGON is detected inPI-PLC treated medium samples. FIG. 13D is a series of photomicrographsfrom an anti-DRAGON immunohistochemical study of adult spinal cord andDRG at low (top) and high (middle) magnification. As a control, theanti-DRAGON antibody was pretreated with the immunogenic DRAGON fragmentprior to immunohistochemical staining (bottom). Scale, 100 μM.

FIGS. 14A-14C are a series of photomicrographs that demonstrate theadhesion of DRG neurons to DRAGON-expressing HEK 293 cells. P14 neonatalDRG neurons were plated on a monolayer of confluent HEK cells (FIG. 14A)and DRAGON transfected HEK cells (FIG. 14B). The culture slides werewashed, fixed, and immunostained for DRG neurons using anti-NeuN(neuronal marker). Double immuno-labeling using anti-NeuN andanti-DRAGON indicates a direct interaction between DRAGON expressing HEKcells and DRG neurons (FIG. 14C). FIG. 14D is a bar graph quantifyingthe adhesion experiment results. A 1.9-fold increase in the number ofadherent DRG neurons when plated on DRAGON-expressing HEK 293 cells,compared to control HEK 293 cells. Pretreatment of the DRAGON-expressingHEK cells with PI-PLC significantly reduced the adherence of DRGneurons.

FIG. 15A is a series of photomicrographs demonstrating the effect ofDRAGON overexpression in Xenopus laevis. Embryos were injected in theanimal pole of 1 out of 2 cells at the 2-cell stage with DRAGON RNA andanalyzed at late neurula (st23) for changes in neural crest patterningand early tadpole stages (st28) for ectopic induction of neural tissue.DRAGON overexpression inhibits twist RNA expression. However, DRAGONinduces ectopic N-tubulin RNA expression. FIG. 15B is a Northern blotfrom animal cap explants demonstrating that DRAGON induces anteriorneural markers, cement gland markers, and nk×2.5 (a cardiac marker).

FIG. 16A is a Northern blot showing the developmental expression ofDRAGON in Zebrafish embryos over the first 36 hours post fertilization(hpf). FIGS. 16B-16C are lateral views of an 18-20 somite stagezebrafish embryo following in situ hybridization using a DRAGONantisense probe (FIG. 16B) or the sense control (FIG. 16C). DRAGONexpression is strongest at the anterior pole and in the tail-bud region(arrows). More diffuse and lower levels of expression are seen in otherparts of the brain. FIG. 16D is a dorsal view of a flat-mounted embryoshowing DRAGON staining in the CNS. DRAGON expression is particularlystrong in the region surrounding the olfactory placodes (black arrow)and in the retina (white arrow). FIGS. 16E-16G are photomicrographsdemonstrating that DRAGON overexpression causes abnormalities in brainmorphology and, at a lower frequency (7-15%), cylopia. FIG. 16H is aNorthern blot of zebrafish embryo RNA demonstrating that a morpholinooligonucleotide (MO) targeted against the splice donor site of DRAGONexon1 blocks RNA splicing and protein expression. An inverted morpholinooligonucleotide (cont. MO), which preserves the base composition, wasused as the control. Primers flanking the intron used for RT-PCR producethe predicted bands from the end products of splicing in the control butnot the experimental morpholinos. FIGS. 161-16J are photomicrographs of24 hour zebrafish embryos following MO injection. Morphologically, theeyes are affected and extensive cell death in the brain obscures theclear definition of the midbrain-hindbrain boundary. FIGS. 16K-16M showTUNEL staining of MO injected embryos at the 21 somite stage revealing apattern of cell death correlating with the pattern of DRAGON expression.

FIG. 17 is a sequence alignment of mDRAGON (SEQ ID NO: 5) and a regionof C. elegans DRAGON. The full length C. elegans DRAGON is also provided(SEQ ID NO: 18).

FIG. 18 is a photomicrograph showing the distribution of DRAGONexpression in the retina and optic nerve of a mouse embryo (E14.5) usingimmunohistochemistry.

FIG. 19 is a photomicrograph showing the distribution of DRAGONexpression in rat glaborous skin (base of the epidermis of the hindpaw)using immunohistochemistry. DRAGON expression is highest in the Merkelcells.

DETAILED DESCRIPTION

DRG11 is a paired homeodomain transcription factor that is expressedboth by dorsal root ganglion (DRG) sensory neurons and by dorsal hornneurons early in development (Saito et al., Mol. Cell. Neurosci.6:280-92, 1995). Its absence, following a null mutation of its gene,leads to abnormalities in the spatio-temporal distribution of sensoryneuron projections to the dorsal horn, as well as defects in dorsal hornmorphogenesis (Chen et al., Neuron 31:59-73, 2001). These developmentalabnormalities may account for a significantly attenuated sensitivity tonoxious stimuli in the DRG11 deficient mice (Chen et al., supra).

DRG11-Responsive Gene Identification

A Genomic Binding Site (GBS) strategy was used in a mouse CpG islandlibrary to isolate genes responsive to the transcription factor DRG11and to identify proteins that are involved in the development of sensorypathways (primary sensory neurons and spinal cord neurons) and otherneurons (FIG. 1A). The general strategy isolates DRG11-binding fragmentsfrom mouse genomic DNA using a fusion protein (DRG-GST) consisting of arecombinant DRG11 DNA binding domain (amino acids 31-90 of mDRG-11) andGST. DRG11-responsive genes are located and isolated from the genomicregion adjacent to the DRG-11 binding site. Mouse CpG islands areselected by the methyl-CpG binding domain of MeCP2, which binds DNAmethylated at CpG and allows fractionation of DNA according to itsdegree of CpG methylation. The CpG library consists of short stretchesof DNA containing a high density of nonmethylated CpG dinucleotides.About 60% of human genes are associated with CpG islands. These regionsoften include the promoter region and one or more exons of associatedgenes, allowing the isolation of full length cDNAs and genomic mapping.

The GBS cloning using the CpG island library was performed according tothe method of Watanabe et al. (Mol. Cell. Biol. 18:442-449, 1998).Briefly, ten micrograms of the mouse library plasmid DNA was incubatedwith the DRG-GST fusion protein. The resulting solution was then passedslowly through a presoaked nitrocellulose filter and washed. The trappedplasmid DNA was eluted from the filter and transformed into DH5α (E.coli) competent cells. The cells were cultured in Luria broth-ampicillin(LB) medium and plasmid DNA was prepared. The cycle was repeated for atotal of three times. After the third cycle, the plasmid DNA library,enriched in genomic fragments that bind to the DRG11 DNA binding domain,was plated on LB-ampicillin agar plates, and individual clones wereamplified, sequenced, and characterized.

Identification and Characterization of Murine DRAGON

Among the most abundant clones obtained, was a 363 base pair (bp) DNAfragment located 750 bp upstream of an open reading frame of a novelgene. Sequence analysis studies indicated that the genomic fragment islocated in the promoter region of the new gene. Genomic databaseanalysis, combined with RT-PCR and RACE (Rapid Amplification of cDNAEnds) of mouse DRG and spinal cord cDNA libraries found that the openreading frame encoded a novel cDNA (SEQ ID NO: 1) that we have calledDRAGON. The nucleotide and predicted 436 amino acid sequence of DRAGON(SEQ ID NO: 5) are shown in (FIG. 1B).

Sequence analysis of the mDRAGON coding region identified conserveddomains with homology to notch-3 (FIG. 5),phosphatidylinositol-4-phosphate-5-kinase type II beta (FIG. 5),insulin-like growth factor binding protein-2 (IGFBP2; FIG. 3),thrombospondin (FIG. 6), ephrin type-B receptor 3 precursor (EPHB3; FIG.4), and Slit-2 (FIG. 6), all of which are known to influence axonalguidance, neurite outgrowth, and other neuronal developmental functions.The C-terminus of mDRAGON is also predicted to contain a hydrophobicdomain indicative of a 21 amino acid extracellular GPI anchoring. Acomputational structure-function analysis of mDRAGON reveals thepresence of a putative signal peptide sequence (FIG. 2), indicating thatthe gene product is a secreted protein, and further supporting anextracellular localization.

Identification of DRAGON Homologs

Sequence homology analysis using a mouse genome database identified twomurine genes homologous to DRAGON. The cDNA sequences of these homologs(mDL-1 and mDL-2) are provided in SEQ ID NO: 2 and SEQ ID NO: 3,respectively. The deduced polypeptide sequences are also provided (SEQID NO: 6 and 7). Sequence alignments indicating areas of homologybetween mDRAGON, mDL-1, and mDL-2 are shown in FIG. 8A. The GPI anchorsequence is predicated to be at the C-terminal 27 and 36 amino acids ofmDL-1 and mDL-2, respectively.

DRG11 Induces DRAGON Expression

Following the initial identification of mDRAGON using GBS cloning, themDRAGON promoter (363 bp fragment) was confirmed to be DRG11-responsiveusing the reporter gene assay generally described by Ogura et al. (Proc.Natl. Acad. Sci. USA, 92:392-396, 1995). The 363 bp fragment wassubcloned into the PGL3-Promoter reporter vector containing an SV40promoter upstream of the luciferase gene. DRG11 triggered a 6-foldincrease in luciferase activity as compared to control (FIG. 7B),revealing the presence of one or several DRG11 response elements in the363 base pair promoter fragment. No induction in luciferase activity wasdetected in the absence of DRG11, indicating that the enhancer activityof the DRAGON promoter fragment was DRG11 dependent.

Tissue Localization of Dragon Gene Expression

In situ hybridization was used to demonstrate that at E12.5 DRG11 andDRAGON expression overlaps (FIG. 7A). In the DRG most neurons expressboth DRG11 and DRAGON; in the spinal cord DRG11 and DRAGON are expressedin the same medial region adjacent to the ventricular zone (FIG. 7A). Apull down assay was carried out to confirm interaction of DRG11 with the363 bp promoter fragment of DRAGON obtained with the GBS screening. Thepromoter fragment was pulled down by a GST-DRG11-DBD fusion protein butnot GST (FIG. 7C). Finally, DRAGON mRNA expression in DRG11 null mutantembryonic mice was examined. DRAGON expression in the spinal cord andDRG were significantly reduced in DRG11−/− mice compared to wildtypelittermates (FIG. 7D).

DRAGON mRNA is expressed in embryonic and adult mouse DRGs, spinal cordand brain, with little or no expression in the liver and kidney, and lowlevels in the heart (FIGS. 7E, 9A, 9D, 9G, 9J, and 10). DRAGONexpression begins early in development (at least E7) (FIG. 7F), muchearlier than DRG11 (E12). Its expression is dynamically regulated in thePNS and CNS during development.

The relative tissue distribution pattern of DRAGON, DL-2 and DL-1 mRNAin mouse embryos (E14.5) indicate that DRAGON and DL-2, but not DL-1,are primarily expressed in the nervous system, and that DRAGON and DL-2expression in the nervous system is largely non-overlapping (FIGS.9G-9L). DRAGON is heavily expressed in DRG neurons and in thedorso-medial mantle layer of the spinal cord, with lower expressionlaterally and ventrally. DL-2 shows no expression in the DRG but strongexpression in the spinal cord and brain. In the spinal cord, DL-2 isexpressed in the midline, extending from the roof to the floor platearound the central canal in the ependymal layer, medial and ventral toDRAGON (FIGS. 9C and 9F). DL-2-expressing neurons are also present inthe marginal layer and ventral horn. A complementary DRAGON and DL-2expression pattern is also present in embryonic brain. DRAGON isexpressed in the alar plate of the myelencephalon, in the marginal layerof the mesencephalon, and with lower intensity laterally, in the basalplate of the pons, and in the cerebellar primordia. DL-2 is expressed inthe ependymal layers of the myelencephalon, mesencephalon and pons. DL-2but not DRAGON is expressed in the telencephalic cortex, most intenselymedially. DRAGON is heavily expressed in the diencephalon, except in theependymal layer where DL-2 is heavily expressed. DRAGON is homogeneouslyexpressed in the striatum whereas DL-2 is only expressed on its medialsurface (FIGS. 9G-9L). DRAGON, but not DL-2, is expressed in the cortexof the future olfactory lobe, retina and olfactory epithelium (FIGS. 11and 12). Both DRAGON and DL-2 are expressed in the cochlea.

The mDL-1 gene has a very specific expression pattern in the developingmouse embryo. Expression was restricted to muscle and cartilage tissuesdistributed along the whole organism, indicating a role in muscle andbone development (FIGS. 9B and 9E). A structure-function analysis of themDL-1 protein sequence indicated the presence of a signal peptidesuggesting that mDL-1, like mDRAGON, is a secreted factor.

DRAGON Protein Expression

A rabbit polyclonal antibody was raised against the peptide sequenceTAAAHSALEDVEALHPRK (SEQ ID NO: 11; residues 388-405 of mDRAGON), presentin the C-terminus of DRAGON, upstream of its hydrophobic tail. Theantibody binds with high affinity to recombinant DRAGON expressed inHEK293T transfected cells, recognizing a band of 50 KDa in Western blots(FIG. 13A). Antibody specificity was confirmed by immunocytochemistry ofDRAGON-expressing HED293T cells (FIG. 13B). Western blots of proteinextracts from neonatal and adult DRG and DRG primary cultures show asimilar band with an additional lower band of 40 KDa, indicatingpossible proteolytic cleavage of endogenous DRAGON. Treatment of HEK293Tcells expressing DRAGON with PI-PLC results in the decrease of DRAGONdetection on HEK cells and its release into the culture medium (FIG.13C), indicating that DRAGON is GPI-anchored.

Immunohistochemistry confirms expression of DRAGON in the DRG, spinalcord and brain in the areas where DRAGON mRNA is found (FIG. 13D). Inthe adult DRG, DRAGON is more abundantly expressed in small neurons withunmyelinated axons than in medium and large myelinated neurons (Aδ andAβ-fibers) (FIG. 13D). In the adult spinal cord, DRAGON expression ismost prominent in the superficial laminae of the dorsal horn (FIG. 13D).Immunohistochemical studies also demonstrated that the DRAGON protein isexpressed in the E14.5 mouse retina and optic nerve (FIG. 18) and skin(FIG. 19).

DRAGON Promotes Cellular Adhesion

Cell surface GPI-anchored proteins, including the ephrins and tenascin,act as neuronal and non-neuronal cell adhesion molecules, binding tomolecules expressed on neighboring cells or in the extracellular matrix.To examine whether DRAGON has a cell adhesion role, we measured theamount of adhesion between DRG neurons and HEK293 cells expressingrecombinant DRAGON. DRAGON expression caused nearly a two-fold increasein the number of cultured DRG neurons that adhered to a monolayer ofDRAGON-expressing HEK cells, compared to control HEK cells (FIGS.14A-14D). Moreover, pretreatment of DRAGON-expressing HEK cells withPI-PLC resulted in only basal levels of DRG adhesion (FIGS. 14A-14D).These results may reflect homophilic or heterophilic DRAGON interactionswith the endogenous DRAGON protein expressed on the surface of DRGneurons.

DRAGON Promotes Neuronal Survival

The anti-DRAGON polyclonal antibody was added to neonatal rat DRGneuronal cultures to investigate the contribution of DRAGON to neuronalsurvival. Neuronal cultures were treated with 0.25% anti-DRAGON serum,0.25% pre-immune serum (negative control), or vehicle. A statisticallysignificant 20-25% increase in neuronal cell death was measuredfollowing anti-DRAGON treatment compared to controls. 0.25% 0.25%Vehicle anti-DRAGON pre-immune Control serum serum (no serum) % viableneurons 41.8% 55.3% 51.8% (mean) Standard Error (S.E.) 1.7% 2.3% 2.5%Number of isolated 12 12 11 DRG cultures (n)Neural Induction in Xenopus Embryos

In order to determine whether DRAGON affects cell differentiation andearly embryonic development, DRAGON was injected into one cell at theanimal pole of Xenopus embryos at the 2-cell stage. Embryos were allowedto develop until early tadpole stages. By injecting one out of twocells, a control side and an experimental side are present in the sameembryo. A variety of markers were measured, including twist (expressedin anterior neural crest cells) and N-tubulin (a general neuronaldifferentiation marker), to determine whether DRAGON affects earlyneural patterning. Ectopic DRAGON caused a decrease in neural crestderivatives, as shown by loss of twist expression (FIG. 15A, top panels)and an increase in neuronal markers (FIG. 15A, bottom panels).

In ectodermal explant assays, DRAGON induced anterior neural markers(FIG. 15B). Nrp1 is a pan-neural marker, Otx2 is expressed within theforebrain and midbrain regions, and XAG is expressed in the cement gland(the most anterior structure in the tadpole). In addition, DRAGONinduced nk×2.5, an early marker of cardiac development.

Identification of Dragon Homologs in Other Species

Zebrafish Dragon Genes

The cDNA and polypeptide sequences of zebrafish homologs of DRAGON (SEQID NO: 25 and 28), DL-1 (SEQ ID NO: 26 and 29), and DL-2 (SEQ ID NO: 27and 30) are provided. The sequence and domain structure of the threezebrafish genes are highly conserved with the mouse genes (70-75%homology) and Northern blot analysis shows a single transcript in eachcase. (FIG. 16A). DRAGON mRNA is present at the 2-4 cell stage ofzebrafish embryogenesis, which is prior to initiation of zygotictranscription, suggesting a maternal or early developmental role for theprotein. After the mid-blastula transition, the levels of DRAGON mRNAincrease and are then maintained at a high level for up to 72 hours postfertilization, the latest stage examined (FIG. 16A).

In-situ hybridization reveals widespread and strong DRAGON expression inthe zebrafish embryo. At the 18-somite stage, DRAGON is expressed alongthe midline in the telencephalon, diencephalon, and mesencephalon (FIGS.16B and 16C). DRAGON is also expressed in the developing retina (FIG.16D).

Overexpression of DRAGON in zebrafish embryos following sense injectioninto the fertilized egg, leads to abnormalities in the morphology of thebrain and eye in 75-85% of treated embryos. The most common featuresinclude abnormal ventricle development, inappropriate cell death,particularly in the hindbrain, and neural tube twisting. DRAGONoverexpression results in cyclopedia in 10-20% of embryos. The singleeye is in an abnormally ventral location, with the anterior portion ofthe brain being dorsal to the eye (FIG. 16E-16G).

Embryos injected with a morpholino antisense oligonucleotide directedagainst the splice-donor site of the first exon of DRAGON show extensiveCNS degeneration with a failure of development of the forebrain,hindbrain, and spinal cord (FIG. 16I-16M). The knockdown of DRAGONsplicing and expression was confirmed by RT-PCR and compared to controls(FIG. 16H). Injected embryos had extensive apoptotic cell death in thebrain, the brainstem and along the entire rostro-caudal extent of thespinal cord, as assessed by TUNEL assay and acridine orange staining. Aninverted control oligonucleotide had no effect.

During early development, DL-1 shows high expression in the notochordand the adjacent adaxial cells (the earliest cells to develop intomuscle fibers). Subsequently, DL-1 is expressed exclusively in thesomites.

DL-2 mRNA first appears during zebrafish development at the three somitestage (approximately 10 hours postfertilization). DL-2 expression peaksat 18 hours, followed by a decrease over the next 72 hours.

Human Dragon Genes

The human homologs of all three murine Dragon gene family have beenidentified using the human genomic Celera database. The alignment of thehuman, mouse, and zebrafish DRAGON proteins is provided in FIG. 8B. Thehuman homologs (SEQ ID NO: 8-10) are about 90% identical to the murineDragon proteins (SEQ ID NO: 5-7).

C. elegans DRAGON

Strong conservation of many domains present in members of the Dragonfamily among different species has also enabled us to identify the C.elegans ortholog (FIG. 17; SEQ ID NO: 18). The strong domainconservation pattern suggests a crucial role in development for thedifferent members of this family.

Identification of Dragon Genes in Other Species

Homologs from other species can easily be identified based on sequenceidentity with the Dragon proteins and nucleic acids disclosed herein.Sequence identity may be measured using sequence analysis software onthe default setting (e.g., Sequence Analysis Software Package of theGenetics Computer Group, University of Wisconsin Biotechnology Center,1710 University Avenue, Madison, Wis. 53705). Such software may matchsimilar sequences by assigning degrees of homology to varioussubstitutions, deletions, and other modifications. Conservativesubstitutions typically include substitutions within the followinggroups: glycine, alanine, valine, isoleucine, leucine; aspartic acid,glutamic acid, asparagine, glutamine; serine, threonine; lysine,arginine; and phenylalanine, tyrosine.

Multiple sequences may also be aligned using the Clustal W(1.4) program(produced by Julie D. Thompson and Toby Gibson of the European MolecularBiology Laboratory, Germany and Desmond Higgins of EuropeanBioinformatics Institute, Cambridge, UK) by setting the pairwisealignment mode to “slow,” the pairwise alignment parameters to includean open gap penalty of 10.0 and an extend gap penalty of 0.1, as well assetting the similarity matrix to “blosum.” In addition, the multiplealignment parameters may include an open gap penalty of 10.0, an extendgap penalty of 0.1, as well as setting the similarity matrix to“blosum,” the delay divergent to 40%, and the gap distance to 8.

In Situ Hybridization

The in situ hybridization methods used herein have been describedpreviously (Karchewski et al., J. Comp. Neurol. 413:327, 1999).Hybridization was performed on fresh frozen, mounted tissue sectionsfrom mouse embryo and adult rat dorsal root ganglia (DRG) usingterminally-labeled oligonucleotide probes. Probes had approximately 50%G-C content and were complementary and selective for mDRAGON mRNAs.Probes were 3′-end labeled with ³⁵S-dATP using a terminal transferasereaction and purified through a spin column (Qiagen). Hybridization wasdone under very high stringency conditions such that probe annealingrequired at least 90% sequence identity (Dagerlind et al.,Histochemistry 98:39, 1992).

Briefly, slides were brought to room-temperature and covered with ahybridization solution (50% formamide, 1× Denhardt's solution, 1%sarcosyl, 10% dextran sulphate, 0.02M phosphate buffer, 4×SSC, 200 nMDTT, 500 mg/ml salmon sperm DNA) containing 10⁷ cpm/ml of labeled probe.Slides were incubated in a humidified chamber at 43° C. for 14-18 hours,then washed 4×15 min in 1×SSC at 55° C. In the final rinse, slides werebrought to room temperature, washed in dH₂O, dehydrated in ethanol, andair dried.

Autoradiograms were generated by dipping slides in NTB2 nuclear trackemulsion and storing in the dark at 4° C. Prior to conventionaldeveloping and fixation, sections were allowed to expose for 1-3 weeks,depending on the abundance of transcript. Unstained tissue was viewedunder darkfield conditions using a fiber-optic darkfield stage adapter(MVI), while stained tissue was examined under brightfield conditions.Control experiments using sense probes were conducted to confirm thespecificity of hybridization. The antisense oligonucleotide probes areas follows: mDRAGON- specific for nucleotides 831-879 of SEQ ID NO:1:(SEQ ID NO:19) 5′-TCG CAC AAA CAC TGT GGT GCC TAT GTA GCG GGC ATG CATCTC TAC GTA-3′. mDL-1- specific for nucleotides 913-960 of SEQ ID NO:2:(SEQ ID NO:20) 5′-CCC AGC TGT CTG TCG AAT GAT GAT AGT TGT TCC AAT GTAGGC AGC TCG-3′ mDL-2- specific for nucleotides 1252-1299 of SEQ ID NO:3:(SEQ ID NO:21) 5′-TTG CCA TCC TCC AAA GCA TAG TAG GCA GCC AGC GTG AAGTTC ACA TCA-3′.

Synthesis of Dragon Proteins

Nucleic acids that encode Dragon family proteins or fragments thereofmay be introduced into various cell types or cell-free systems forexpression, thereby allowing purification of these Dragon proteins forbiochemical characterization, large-scale production, antibodyproduction, and patient therapy.

Eukaryotic and prokaryotic Dragon expression systems may be generated inwhich a Dragon family gene sequence is introduced into a plasmid orother vector, which is then used to transform living cells. Constructsin which the Dragon cDNA contains the entire open reading frame insertedin the correct orientation into an expression plasmid may be used forprotein expression. Alternatively, portions of the Dragon genesequences, including wild-type or mutant Dragon sequences, may beinserted. Prokaryotic and eukaryotic expression systems allow variousimportant functional domains of the Dragon proteins to be recovered, ifdesired, as fusion proteins, and then used for binding, structural, andfunctional studies and also for the generation of appropriateantibodies.

Typical expression vectors contain promoters that direct the synthesisof large amounts of mRNA corresponding to the inserted Dragon nucleicacid in the plasmid-bearing cells. They may also include a eukaryotic orprokaryotic origin of replication sequence allowing for their autonomousreplication within the host organism, sequences that encode genetictraits that allow vector-containing cells to be selected for in thepresence of otherwise toxic drugs, and sequences that increase theefficiency with which the synthesized mRNA is translated. Stablelong-term vectors may be maintained as freely replicating entities byusing regulatory elements of, for example, viruses (e.g., the OriPsequences from the Epstein Barr Virus genome). Cell lines may also beproduced that have integrated the vector into the genomic DNA, and inthis manner the gene product is produced on a continuous basis.

Expression of foreign sequences in bacteria, such as Escherichia coli,requires the insertion of the Dragon nucleic acid sequence into abacterial expression vector. Such plasmid vectors contain severalelements required for the propagation of the plasmid in bacteria, andfor expression of the DNA inserted into the plasmid. Propagation of onlyplasmid-bearing bacteria is achieved by introducing, into the plasmid,selectable marker-encoding sequences that allow plasmid-bearing bacteriato grow in the presence of otherwise toxic drugs. The plasmid alsocontains a transcriptional promoter capable of producing large amountsof mRNA from the cloned gene. Such promoters may be (but are notnecessarily) inducible promoters that initiate transcription uponinduction. The plasmid also preferably contains a polylinker to simplifyinsertion of the gene in the correct orientation within the vector.

Mammalian cells can also be used to express a Dragon protein. Stable ortransient cell line clones can be made using Dragon expression vectorsto produce Dragon proteins in a soluble (truncated and tagged) ormembrane anchored (native) form. Appropriate cell lines include, forexample, COS, HEK293T, CHO, or NIH cell lines.

Once the appropriate expression vectors containing a Dragon gene,fragment, fusion, or mutant are constructed, they are introduced into anappropriate host cell by transformation techniques, such as, but notlimited to, calcium phosphate transfection, DEAE-dextran transfection,electroporation, microinjection, protoplast fusion, or liposome-mediatedtransfection. The host cells that are transfected with the vectors ofthis invention may include (but are not limited to) E. coli or otherbacteria, yeast, fungi, insect cells (using, for example, baculoviralvectors for expression in SF9 insect cells), or cells derived from mice,humans, or other animals. In vitro expression of Dragon proteins,fusions, polypeptide fragments, or mutants encoded by cloned DNA mayalso be used. Those skilled in the art of molecular biology willunderstand that a wide variety of expression systems and purificationsystems may be used to produce recombinant Dragon proteins and fragmentsthereof. Some of these systems are described, for example, in Ausubel etal. (supra).

Once a recombinant protein is expressed, it can be isolated from celllysates using protein purification techniques such as affinitychromatography. Once isolated, the recombinant protein can, if desired,be purified further by e.g., by high performance liquid chromatography(HPLC; e.g., see Fisher, Laboratory Techniques In Biochemistry AndMolecular Biology, Work and Burdon, Eds., Elsevier, 1980).

Polypeptides of the invention, particularly short Dragon fragments canalso be produced by chemical synthesis (e.g., by the methods describedin Solid Phase Peptide Synthesis, 2nd ed., 1984, The Pierce ChemicalCo., Rockford, Ill.).

Dragon Fusion Proteins

Also included in the invention are Dragon family proteins fused toheterologous sequences, such as detectable markers (for example,proteins that may be detected directly or indirectly such as greenfluorescent protein, hemagglutinin, or alkaline phosphatase), DNAbinding domains (for example, GAL4 or LexA), gene activation domains(for example, GAL4 or VP16), purification tags, or secretion signalpeptides. These fusion proteins may be produced by any standard method.For production of stable cell lines expressing a Dragon fusion protein,PCR-amplified Dragon nucleic acids may be cloned into the restrictionsite of a derivative of a mammalian expression vector. For example, KA,which is a derivative of pcDNA3 (Invitrogen, Carlsbad, Calif.) containsa DNA fragment encoding an influenza virus hemagglutinin (HA).Alternatively, vector derivatives encoding other tags, such as c-myc orpoly Histidine tags, can be used.

The Dragon expression construct may be co-transfected, with a markerplasmid, into an appropriate mammalian cell line (e.g. COS, HEK293T, orNIH 3T3 cells) using, for example, Lipofectamine™ (Gibco-BRL,Gaithersburg, Md.) according to the manufacturer's instructions, or anyother suitable transfection technique known in the art. Suitabletransfection markers include, for example, P-galactosidase or greenfluorescent protein (GFP) expression plasmids or any plasmid that doesnot contain the same detectable marker as the Dragon fusion protein. TheDragon-expressing cells can be sorted and further cultured, or thetagged Dragon can be purified.

In one particular example, a DRAGON open reading frame (ORF) wasamplified by polymerase chain reaction (PCR) using standard techniquesand primers containing restriction sites (e.g. Sal I sites). The topstrand primer consisted of the sequence 5′-ATA AGC TTA TGG GCG TGA GAGCAG CAC CTT CC-3′ (SEQ ID NO: 22) and the bottom strand primer consistedof the sequence 5′-GAA GTC GAC GAA ACA ACT CCT ACA AAA AC-3′ (SEQ ID NO:23). DRAGON cDNA was also amplified without the signal peptide andsubcloned into a vector (pSecTagHis) having a strong secretion signalpeptide. The same bottom strand primer was used (SEQ ID NO: 23);however, the top strand primer was substituted for one having thesequence 5′-CTC AAG CTT CAG CCT ACT CAA TGC CGA ATC-3′ (SEQ ID NO: 24).

In another example, we generated DRAGON-alkaline phosphatase (AP) fusionprotein using the mammalian expression vector, pAPtag-5′ (Flanagan etal., Meth. Enzymol. 327:198-210, 2000). When expressed in mammaliancells (e.g. HEK 293), the DRAGON-AP fusion protein is secreted at highlevels into the culture medium and is easily detected by the AP activityassay. The resulting DRAGON-AP fusion protein can be used to screenexpression libraries to identify, clone, sequence, and characterizemolecules which interact with DRAGON, such as cell surface receptors orendogenous DRAGON ligands. Of course, this method is broadly applicableto all Dragon-family proteins and can be used in conjunction with anynumber of suitable tags known in the art.

Interaction Trap Assays

Two-hybrid methods, and modifications thereof, may also be used toidentify novel proteins that interact with Dragon-family proteins, andhence may be naturally occurring Dragon ligands or receptors. Inaddition, regulators of Dragon, e.g., proteins that interfere with orenhance the interaction between Dragon and other proteins, may beidentified by the use of a three-hybrid system. Such assays arewell-known to skilled artisans, and may be found, for example, inAusubel et al. (supra).

Generation of Anti-Dragon Antibodies

In order to prepare polyclonal antibodies, Dragon family proteins,fragments, or fusion proteins containing defined portions of Dragonproteins may be synthesized in bacterial, fungal, or mammalian cells byexpression of corresponding DNA sequences in a suitable cloning vehicle.The proteins can be purified, coupled to a carrier protein, mixed withFreund's adjuvant (to enhance stimulation of the antigenic response inan innoculated animal), and injected into rabbits or other laboratoryanimals. Following booster injections at bi-weekly intervals, therabbits or other laboratory animals are then bled and the sera isolated.The sera can be used directly or can be purified prior to use by variousmethods, including affinity chromatography employing reagents such asProtein A-Sepharose, antigen-Sepharose, and anti-mouse-Ig-Sepharose. Thesera can then be used to probe protein extracts from Dragon-expressingtissue electrophoretically fractionated on a polyacrylamide gel toidentify Dragon proteins. Alternatively, synthetic peptides can be madethat correspond to the antigenic portions of the protein and used toinnoculate the animals. As described above, a polyclonal antibodyagainst mDRAGON was created using, as the immunogenic DRAGON fragment, apolypeptide corresponding to residues 388-405 of SEQ ID NO: 5. Suitableimmunogens for creating anti-hDRAGON antibodies include, for example,the polypeptide sequences encoded by residues 54-72, 277-294, or 385-408of SEQ ID NO: 8.

Alternatively, monoclonal antibodies may be prepared using Dragonproteins described above and standard hybridoma technology (see, e.g.,Kohler et al., Nature 256:495, 1975; Kohler et al., Eur. J. Immunol.6:511, 1976; Kohler et al., Eur. J. Immunol. 6:292, 1976; Hammerling etal., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, New York,N.Y., 1981). Once produced, monoclonal antibodies are also tested forspecific Dragon protein recognition by Western blot orimmunoprecipitation analysis.

Antibodies of the invention may also be produced using Dragon amino acidsequences that do not reside within highly conserved regions, and thatappear likely to be antigenic, as analyzed by criteria such as thoseprovided by the Peptide Structure Program (Genetics Computer GroupSequence Analysis Package, Program Manual for the GCG Package, Version7, 1991) using the algorithm of Jameson and Wolf (CABIOS 4:181, 1988).

Use of Dragon Proteins and Nucleic Acids in Diagnosis

Dragon family proteins may be used in diagnosing existing disorders orthe propensity for developing disorders of the nervous system (DRAGONand DL-2) or bone, muscle, skin, joint, and cartilage tissue (DL-1),where a decrease or increase in the level of Dragon protein or nucleicacid production, relative to a control, provides an indication of adeleterious condition. Alternatively, a patient sample may be analyzedfor one or more alterations in a Dragon nucleic acid sequence, comparedto a wild-type Dragon sequence, using a mismatch detection approach. Thealteration in the Dragon sequence need not be in a coding region.Alterations in, for example, promoter regions can result in alterationsof Dragon protein levels and/or tissue distribution. Wild-type Dragonnucleic acid sequences for use in this assay include SEQ ID NO: 1-4 and31-32.

Generally, these techniques involve PCR amplification of nucleic acidfrom the patient sample, followed by identification of the mutation(e.g., mismatch) by either altered hybridization, aberrantelectrophoretic gel migration, binding or cleavage mediated by mismatchbinding proteins, or direct nucleic acid sequencing. Any of thesetechniques may be used to facilitate mutant Dragon detection, and eachis well known in the art (see, for example, Orita et al., Proc. Natl.Acad. Sci. USA 86:2766-2770, 1989; and Sheffield et al., Proc. Natl.Acad. Sci. USA 86:232-236, 1989).

Mismatch detection assays may be used to diagnose a Dragon nucleicacid-mediated predisposition to a nervous system, bone, muscle, skin orcartilage condition. For example, a patient heterozygous for a Dragonmutation may show no clinical symptoms and yet possess a higher thannormal probability of developing one or more types of these diseases.Given this diagnosis, a patient may take precautions to control theirexposure to adverse environmental factors and to carefully monitor theirmedical condition (for example, through frequent physical examinations).This type of Dragon diagnostic approach may also be used to detectDragon nucleic acid mutations in prenatal screens.

Measurement of Dragon RNA is also a useful diagnostic. For example, adecrease in a Dragon mRNA or protein in a subject, relative to a controlsubject, would suggest a diagnosis of the presence or propensity foracquiring a disorder of the nervous system, or the bone, muscle, skin,or joint tissue. In addition, a decrease in Dragon mRNA or protein,relative to control, may correlate with a poor prognosis for treatmentof these conditions using a non-Dragon therapy.

Levels of Dragon protein or nucleic acid expression may be assayed byany standard technique and compared to control samples showing normalDragon protein or nucleic acid expression. For example, expression in abiological sample (e.g., a biopsy) may be monitored by standard Northernblot analysis, using, for example, probes designed from a Dragon nucleicacid. Measurement of such expression may be aided by PCR (see, e.g.,Ausubel et al., supra; PCR Technology: Principles and Applications forDNA Amplification, ed., H. A. Ehrlich, Stockton Press, NY; and Yap andMcGee, Nucl. Acids Res. 19:4294, 1991).

In yet another approach, immunoassays may be used to detect or monitor aDragon protein in a biological sample. Dragon-specific polyclonal ormonoclonal antibodies may be used in any standard immunoassay format(e.g., ELISA, Western blot, or RIA assay) to measure Dragon levels;again comparison is to wild-type Dragon levels. Examples of immunoassaysare described, e.g., in Ausubel et al. (supra). Immunohistochemicaltechniques may also be utilized for Dragon detection. For example, atissue sample may be obtained from a patient, and a section stained forthe presence of a Dragon protein using an antibody against that proteinand any standard detection system (e.g., one which includes a secondaryantibody conjugated to horseradish peroxidase). General guidanceregarding such techniques can be found in, e.g., Bancroft and Stevens(Theory and Practice of Histological Techniques, Churchill Livingstone,1982) and Ausubel et al. (supra).

Identification of Candidate Compounds for Treatment of Dragon-relatedConditions

A candidate compound that is beneficial in the treatment, stabilization,or prevention of a Dragon-related condition (e.g. disorders of thenervous system, retina, skin, and bone, muscle, joint, or cartilagetissue) can be identified by the methods of the present invention. Acandidate compound can be identified for its ability to affect thebiological activity of a Dragon protein or the expression of a Dragongene or to mimic its action. Compounds that are identified by themethods of the present invention that increase the biological activityor expression levels of a Dragon protein or that compensate for the lossof Dragon protein activity or gene expression, for example, due to lossof the Dragon gene due to a genetic lesion, can be used in the treatmentor prevention of a Dragon-related condition. A candidate compoundidentified by the present invention can mimic the biological activity ofa Dragon protein, bind a Dragon protein, modulate (e.g., increase ordecrease) transcription of a Dragon gene, or modulate translation of aDragon mRNA.

Any number of methods are available for carrying out screening assays toidentify new candidate compounds that promote the expression of a Dragongene. In one working example, candidate compounds are added at varyingconcentrations to the culture medium of cultured cells expressing one ofthe Dragon nucleic acid sequences of the invention. Gene expression isthen measured, for example, by microarray analysis, Northern blotanalysis (Ausubel et al., supra), or RT-PCR, using any appropriatefragment prepared from the nucleic acid molecule as a hybridizationprobe. The level of Dragon gene expression in the presence of thecandidate compound is compared to the level measured in a controlculture medium lacking the candidate compound. A compound which promotesan increase in the expression of a Dragon gene is considered useful inthe invention and may be used as a therapeutic to treat a human patient.

In another working example, the effect of candidate compounds may bemeasured at the level of Dragon protein production using the samegeneral approach and standard immunological techniques, such as Westernblotting or immunoprecipitation with an antibody specific for a Dragonprotein. For example, immunoassays may be used to detect or monitor theexpression of at least one of the polypeptides of the invention in anorganism. Polyclonal or monoclonal antibodies that are capable ofbinding to a Dragon protein may be used in any standard immunoassayformat (e.g., ELISA, Western blot, or RIA assay) to measure the level ofthe protein. In some embodiments, a compound that promotes an increasein Dragon expression or biological activity is considered particularlyuseful.

Expression of a reporter gene that is operably linked to a Dragonpromoter can also be used to identify a candidate compound for treatingor preventing a Dragon-related condition. Assays employing the detectionof reporter gene products are extremely sensitive and readily amenableto automation, hence making them ideal for the design of high-throughputscreens. Assays for reporter genes may employ, for example,calorimetric, chemiluminescent, or fluorometric detection of reportergene products. Many varieties of plasmid and viral vectors containingreporter gene cassettes are easily obtained. Such vectors containcassettes encoding reporter genes such as lacZ/β-galactosidase, greenfluorescent protein, and luciferase, among others. A genomic DNAfragment carrying a Dragon-specific transcriptional control region(e.g., a promoter and/or enhancer) is first cloned using standardapproaches (such as those described by Ausubel et al. (supra). The DNAcarrying the Dragon transcriptional control region is then inserted, byDNA subcloning, into a reporter vector, thereby placing a vector-encodedreporter gene under the control of the Dragon transcriptional controlregion. The activity of the Dragon transcriptional control regionoperably linked to the reporter gene can then be directly observed andquantified as a function of reporter gene activity in a reporter geneassay.

In one embodiment, for example, the Dragon transcriptional controlregion could be cloned upstream from a luciferase reporter gene within areporter vector. This could be introduced into the test cells, alongwith an internal control reporter vector (e.g., a lacZ gene under thetranscriptional regulation of the β-actin promoter). After the cells areexposed to the test compounds, reporter gene activity is measured andDragon reporter gene activity is normalized to internal control reportergene activity.

In addition, candidate compounds may be identified using any of theDragon fusion proteins described above (e.g., as compounds that bind tothose fusion proteins), or by any of the two-hybrid or three-hybridassays described above.

A candidate compound identified by the methods of the present inventioncan be from natural as well as synthetic sources. Those skilled in thefield of drug discovery and development will understand that the precisesource of test extracts or compounds is not critical to the methods ofthe invention. Examples of such extracts or compounds include, but arenot limited to, plant-, fungal-, prokaryotic-, or animal-based extracts,fermentation broths, and synthetic compounds, as well as modification ofexisting compounds. Numerous methods are also available for generatingrandom or directed synthesis (e.g., semi-synthesis or total synthesis)of any number of chemical compounds, including, but not limited to,saccharide-, lipid-, peptide-, and nucleic acid-based compounds.Synthetic compound libraries are commercially available from BrandonAssociates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee, Wis.).Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant, and animal extracts are commercially available from anumber of sources, including Biotics (Sussex, UK), Xenova (Slough, UK),Harbor Branch Oceangraphics Institute (Ft. Pierce, Fla.), and PharmaMar,U.S.A. (Cambridge, Mass.). In addition, natural and syntheticallyproduced libraries are produced, if desired, according to methods knownin the art, e.g., by standard extraction and fractionation methods.Furthermore, if desired, any library or compound is readily modifiedusing standard chemical, physical, or biochemical methods.

Use of Transgenic Animals to Identify a Candidate Compound

The present invention also provides methods for using transgenic andknockout animals that develop a Dragon-related condition and accuratelyrecapitulate many of the features of the Dragon-related conditionassociated with loss or mutation of a Dragon gene. Desirably, the Dragongene is used to produce the transgenic animal or the Dragon gene is thetarget of the knockout. However, other genes involved in or related toDragon expression or activity, may also be used to produce transgenicanimals so that the effect on a Dragon-related condition may be studiedin this context.

A transgenic animal expressing a mutant Dragon gene can be used toidentify candidate compounds that are useful for the treatment orprevention of a Dragon-related condition. Transgenic animals expressinga conditional mutant Dragon gene (e.g., using a tetracycline regulatablesystem) can also be generated by methods well known to those skilled inthe art; such methods are described in, for example, WO 94/29442, WO96/40892, WO 96/01313, and Yamamoto et al. (Cell 101:57-66, 2000). Inaddition, the knockout animal may be a conditional knockout using, forexample, the FLP/FRT system described in, for example, U.S. Pat. No.5,527,695, and in Lyznik et al. (Nucleic Acid Research 24:3784-3789,1996) or the Cre-lox recombination system described, for example, inKilby et al. (Trends in Genetics 9:413-421, 1993).

Transgenic animals may be made using standard techniques, such as thosedescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory, N.Y., 1989). Any tissue specific promotermay direct the expression of any Dragon protein used in the invention,such as neuron-specific promoters, muscle-specific promoters,skin-specific promoters, retina-specific promoters, and bone-specificpromoters.

The disclosed transgenic and knock-out animals may be used as researchtools to determine genetic and physiological features of aDragon-related condition, and for identifying compounds that can affectsuch conditions. Knockout animals also include animals where the normalDragon gene(s) has been inactivated or removed and replaced with apolymorphic allele of this gene. These animals can serve as a modelsystem for the risk of developing, treating, stabilizing, or preventinga Dragon-related condition that is associated with a Dragon genepolymorphism or mutation.

In general, a transgenic or knockout animal can be used to identify acandidate compound useful for treating or preventing a Dragon-relatedcondition by contacting the transgenic or knockout animal with thecandidate compound and comparing the presence, absence, or level ofexpression of genes, either at the RNA level or at the protein level, intissue from a transgenic or knockout animal as described above, andtissue from a matching non-transgenic or knockout animal. Standardtechniques for detecting RNA expression, e.g., by Northern blotting, orprotein expression, e.g., by Western blotting, are well known in theart. The response to or progression of disease in a transgenic orknockout animal, as compared with non-transgenic or knockout animals canbe used to identify compounds that may be effective therapeutics againsta Dragon-related condition, such as nervous system disorders ordisorders of muscle, skin, bone, or cartilage tissue. Transgenic andknockout animals can also be used to predict whether compoundsidentified as therapeutics will affect disease progression.

Any transgenic animal, or cells derived from these animals, may beconstructed and used for compound screening. Preferable animal modelsinclude, without limitation, mice, rats, rabbits, and flies.

Regulation of Stem Cell Fate Using Dragon Family Proteins

Differentiation of stem cells, particularly ES cells, can beaccomplished by exposing the cells to supraphysiological concentrationsof Dragon proteins. Specifically, DRAGON or DL-2 can induce a stem cellto adopt a neuronal phenotype, whereas DL-1 promotes myogenicphenotypes. Stem cell differentiation may be accomplished using anyappropriate technique. For example, transgenic stem cells overexpressinga Dragon protein can be created. Preferably, the Dragon gene is operablylinked to an inducible promoter in order to control the timing and levelof Dragon protein expression. Alternatively, stem cell differentiationcan be done by the treatment with an exogenous Dragon protein.Typically, a recombinant Dragon protein is produced using a non-stemcell line (e.g., CHO cells), the Dragon protein is isolated, and thestem cells are treated in vitro with the protein.

Dragon-induced stem cell differentiation into neuronal phenotypes can befacilitated by blocking competing differentiation pathways (e.g.,pathways that lead to differentiation into cell types of mesodermal orendodermal origin). Examples of these competing pathways include, butare not limited to, signaling pathways for TGF-β superfamily members(Nodal, Activin, and bone morphogenic proteins (BMPs) 2, 4, and 7),which have been shown to be important for endoderm and mesodermdifferentiation (Nature Reviews Neurosci. 3: 271-280). By inhibiting anyone of these TGF-β family members that lead to endoderm or mesodermdifferentiation, differentiation into a cell of neuroectodermal originis favored.

It will be apparent to one of skill in the art that the timing andextent of TGF-β pathway inhibition and overexpression or application ofa Dragon protein will vary depending on the methods and dosages used.For example, inhibition of a of TGF-β pathway by gene knockouttechnology persists throughout the lifetime of an ES cell, whereasinhibition of the same pathway via antisense oligonucleotides isgenerally transient such that antisense oligonucleotides need to bereapplied. In the latter case, one of skill in the art would be able toreadily determine when and how much of the antisense oligonucleotide toreapply to promote neuronal differentiation.

Stem cells that have been induced to differentiate along a neuronal(using DRAGON or DL-2) or myogenic (using DL-1) lineage can betransplanted into a patient in need of cell replacement therapy. Forexample, patients diagnosed as having neurodegenerative diseases (e.g.,Alzheimer's disease, Parkinson's disease, and Huntington's disease) canbe treated by transplanting, into affected brain regions, stem cellsthat have been induced to differentiate along a neuronal lineage byexposure to DRAGON or DL-2. Stem cells treated with DL-1 to inducemyogenic differentiation can be transplanted into patients diagnosed ashaving a muscle wasting disease such as muscular dystrophy, myotoniacongenital, or myotonic dystrophy.

Administration of a Dragon Protein or a Candidate Compound for theTreatment or Prevention of a Dragon-Related Condition

The present invention also includes the administration of a Dragonfamily protein for the treatment or prevention of a Dragon-relatedcondition. The administration of a biologically active Dragon proteinthat, regardless of its method of manufacture, retains full biologicalactivity, can be envisioned as restoring Dragon biological activity in apatient lacking endogenous activity of a Dragon protein due to a loss orreduction of expression or biological activity, e.g., by mutation orloss of a Dragon gene or cells that normally express a Dragon.

Peptide agents of the invention, such as a Dragon protein, can beadministered to a subject, e.g., a human, directly or in combinationwith any pharmaceutically acceptable carrier or salt known in the art.Pharmaceutically acceptable salts may include non-toxic acid additionsalts or metal complexes that are commonly used in the pharmaceuticalindustry. Examples of acid addition salts include organic acids such asacetic, lactic, pamoic, maleic, citric, malic, ascorbic, succinic,benzoic, palmitic, suberic, salicylic, tartaric, methanesulfonic,toluenesulfonic, or trifluoroacetic acids or the like; polymeric acidssuch as tannic acid, carboxymethyl cellulose, or the like; and inorganicacids such as hydrochloric acid, hydrobromic acid, sulfuric acidphosphoric acid, or the like. Metal complexes include zinc, iron, andthe like. One exemplary pharmaceutically acceptable carrier isphysiological saline. Other physiologically acceptable carriers andtheir formulations are known to one skilled in the art and described,for example, in Remington's Pharmaceutical Sciences, (19th edition), ed.A. Gennaro, 1995, Mack Publishing Company, Easton, Pa.

Pharmaceutical formulations of a therapeutically effective amount of apeptide agent or candidate compound of the invention, orpharmaceutically acceptable salt-thereof, can be administered orally,parenterally (e.g. intramuscular, intraperitoneal, intravenous, orsubcutaneous injection), or by intrathecal or intracerebroventricularinjection in an admixture with a pharmaceutically acceptable carrieradapted for the route of administration.

Methods well known in the art for making formulations are found, forexample, in Remington's Pharmaceutical Sciences (19th edition), ed. A.Gennaro, 1995, Mack Publishing Company, Easton, Pa. Compositionsintended for oral use may be prepared in solid or liquid forms accordingto any method known to the art for the manufacture of pharmaceuticalcompositions. The compositions may optionally contain sweetening,flavoring, coloring, perfuming, and/or preserving agents in order toprovide a more palatable preparation. Solid dosage forms for oraladministration include capsules, tablets, pills, powders, and granules.In such solid forms, the active compound is admixed with at least oneinert pharmaceutically acceptable carrier or excipient. These mayinclude, for example, inert diluents, such as calcium carbonate, sodiumcarbonate, lactose, sucrose, starch, calcium phosphate, sodiumphosphate, or kaolin. Binding agents, buffering agents, and/orlubricating agents (e.g., magnesium stearate) may also be used. Tabletsand pills can additionally be prepared with enteric coatings.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and soft gelatincapsules. These forms contain inert diluents commonly used in the art,such as water or an oil medium. Besides such inert diluents,compositions can also include adjuvants, such as wetting agents,emulsifying agents, and suspending agents.

Formulations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, or emulsions. Examples of suitablevehicles include propylene glycol, polyethylene glycol, vegetable oils,gelatin, hydrogenated naphalenes, and injectable organic esters, such asethyl oleate. Such formulations may also contain adjuvants, such aspreserving, wetting, emulsifying, and dispersing agents. Biocompatible,biodegradable lactide polymer, lactide/glycolide copolymer, orpolyoxyethylene-polyoxypropylene copolymers may be used to control therelease of the compounds. Other potentially useful parenteral deliverysystems for the proteins of the invention include ethylene-vinyl acetatecopolymer particles, osmotic pumps, implantable infusion systems, andliposomes.

Liquid formulations can be sterilized by, for example, filtrationthrough a bacteria-retaining filter, by incorporating sterilizing agentsinto the compositions, or by irradiating or heating the compositions.Alternatively, they can also be manufactured in the form of sterile,solid compositions which can be dissolved in sterile water or some othersterile injectable medium immediately before use.

The amount of active ingredient in the compositions of the invention canbe varied. One skilled in the art will appreciate that the exactindividual dosages may be adjusted somewhat depending upon a variety offactors, including the protein being administered, the time ofadministration, the route of administration, the nature of theformulation, the rate of excretion, the nature of the subject'sconditions, and the age, weight, health, and gender of the patient.Generally, dosage levels of between 0.1 μg/kg to 100 mg/kg of bodyweight are administered daily as a single dose or divided into multipledoses. Desirably, the general dosage range is between 250 μg/kg to 5.0mg/kg of body weight per day. Wide variations in the needed dosage areto be expected in view of the differing efficiencies of the variousroutes of administration. For instance, oral administration generallywould be expected to require higher dosage levels than administration byintravenous injection. Variations in these dosage levels can be adjustedusing standard empirical routines for optimization, which are well knownin the art. In general, the precise therapeutically effective dosagewill be determined by the attending physician in consideration of theabove identified factors.

The protein or candidate compound of the invention can be administeredin a sustained release composition, such as those described in, forexample, U.S. Pat. No. 5,672,659 and U.S. Pat. No. 5,595,760. The use ofimmediate or sustained release compositions depends on the type ofcondition being treated. If the condition consists of an acute orsubacute disorder, a treatment with an immediate release form will bepreferred over a prolonged release composition. Alternatively, forpreventative or long-term treatments, a sustained released compositionwill generally be preferred.

The protein or candidate compound of the present invention can beprepared in any suitable manner. The protein or candidate compound canbe isolated from naturally occurring sources, recombinantly produced, orproduced synthetically, or produced by a combination of these methods.The synthesis of short peptides is well known in the art. See e.g.Stewart et al., Solid Phase Peptide Synthesis (Pierce Chemical Co., 2ded., 1984).

Gene Therapy

Another example of how Dragon family polynucleotides of the inventioncan be effectively used in treatment is gene therapy. See, generally,for example, U.S. Pat. No. 5,399,346. The general principle is tointroduce the polynucleotide, for example, a Dragon gene, into a targetcell in a patient, and allow it to supplement the activity of thedefective endogenous Dragon protein. Alternatively, a Dragon gene can beinserted into an embryonic or adult stem or progenitor cell to promotecell survival or induce differentiation into a particular cell fate.

Entry into the cell is facilitated by suitable techniques known in theart such as providing the polynucleotide in the form of a suitablevector, or encapsulation of the polynucleotide in a liposome.

A desired mode of gene therapy is to provide the polynucleotide in sucha way that it will replicate inside the cell, enhancing and prolongingthe desired effect. Thus, the polynucleotide is operably linked to asuitable promoter, such as the natural promoter of the correspondinggene, a heterologous promoter that is intrinsically active in neuronal,bone, muscle, skin, joint, or cartilage cells, or a heterologouspromoter that can be induced by a suitable agent.

Other Embodiments

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. Although the foregoing invention has beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, it will be readily apparent to those ofordinary skill in the art in light of the teachings of this inventionthat certain changes and modifications may be made thereto withoutdeparting from the spirit or scope of the appended claims. Otherembodiments are within the claims.

1. A vector comprising a polynucleotide sequence encoding a polypeptidehaving an amino acid sequence at least 85% identical to a fragment ofhuman DRAGON-like-2 (DL-2) (SEQ ID NO: 10).
 2. The vector of claim 1,wherein said fragment of human DL-2 is at least 50 amino acids.
 3. Thevector of claim 2, wherein said polypeptide has 100% sequence identityto said fragment of human DL-2.
 4. The vector of claim 1, wherein saidfragment of DL-2 lacks the N-terminal signal sequence, the C-terminalanchor sequence, or both.
 5. A method for producing a recombinant humanDL-2 fragment comprising: a) introducing into a cell the vector of claim1; and b) culturing said cell under conditions where said recombinanthuman DL-2 fragment is expressed.
 6. The method of claim 5 furthercomprising purifying said recombinant human DL-2 fragment of step (b).7. A substantially pure polypeptide comprising an amino acid sequence atleast 85% identical to the sequence of human DRAGON-like-2 (DL-2) (SEQID NO: 10).
 8. The polypeptide of claim 7 having at least one activityselected from the group consisting of: promotion of cellular adhesion,promotion of neuronal survival, and neural induction.
 9. The polypeptideof claim 7, wherein said polypeptide lacks the N-terminal signalsequence, the C-terminal GPI anchor domain, or both.
 10. The polypeptideof claim 7, wherein said sequence is at least 95% identical to thesequence of human DL-2.
 11. The polypeptide of claim 7, wherein saidsequence is at least 99% identical to the sequence of human DL-2. 12.The polypeptide of claim 7, wherein said sequence is 100% identical tothe sequence of human DL-2.
 13. The polypeptide of claim 7, wherein saidpolypeptide is recombinant.
 14. A pharmaceutical composition comprisingthe polypeptide of claim 7 and a pharmaceutically acceptable carrier.15. A substantially pure fusion protein comprising the polypeptide ofclaim 7 fused to a heterologous sequence.
 16. A substantially purepolypeptide comprising an amino acid sequence at least 85% identical toa fragment of human DL-2 (SEQ ID NO: 10).
 17. The polypeptide of claim16 having at least one activity selected from the group consisting of:promotion of cellular adhesion, promotion of neuronal survival, andneural induction.
 18. The polypeptide of claim 16, wherein said sequenceis at least 95% identical to the sequence of human DL-2.
 19. Thepolypeptide of claim 16, wherein said sequence is at least 99% identicalto the sequence of human DL-2.
 20. The polypeptide of claim 16, whereinsaid sequence is 100% identical to the sequence of human DL-2.
 21. Thepolypeptide of claim 16, wherein said fragment is at least 50 aminoacids.
 22. The polypeptide of claim 16, wherein said fragment lacks theN-terminal signal sequence, the C-terminal anchor sequence, or both. 23.The polypeptide of claim 16 or 22, wherein said fragment is at least 400amino acids.
 24. A pharmaceutical composition comprising the polypeptideof claim 16 and a pharmaceutically acceptable carrier.
 25. Apharmaceutical composition comprising the polypeptide of claim 23 and apharmaceutically acceptable carrier.
 26. A substantially pure fusionprotein comprising the polypeptide of claim 16 fused to a heterologoussequence.
 27. The fusion protein of claim 26, wherein said fragment isat least 50 amino acids.
 28. The fusion protein of claim 26, whereinsaid fragment is at least 400 amino acids.
 29. A pharmaceuticalcomposition comprising the fusion protein of claim 26 and apharmaceutically acceptable carrier.
 30. The fusion protein of claim 27or 28, wherein said fragment lacks the N-terminal signal sequence, theC-terminal anchor sequence, or both.
 31. A pharmaceutical compositioncomprising the fusion protein of claim 30 and a pharmaceuticallyacceptable carrier.